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ROS production
Laboratory protocolTo determine ROS production we used the modified version by Choi et al. (2006) of the microscopic Nitroblue tetrazolium (NBT, Sigma-Aldrich) assay. Briefly, DCs were stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT, which allows the precipitation of formazan particles in presence of ROS. Blue formazan particles were dissolved using 2M KOH and DMSO and its absorbance was measured using a microplate reader at 620 nm. The absorbance of dissolved NBT increased in proportion to cell number, incubation time, and stimulus concentration.
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DC stimulation with spores or yeasts
DCs are stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT.
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Dissolve blue formazan particles
Blue formazan particles are dissolved using 2M KOH and DMSO.
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Measuring of absorbance
Absorbance is measured using a microplate reader at 620 nm.
created over 15 years ago
(21 January 2010)
last modified over 13 years ago
(28 September 2011)
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